Transduction efficiency and RNA packaging by alternative Psi packaging sequence in various transgene vectors.

<p>A) Various transgene plasmids harboring SW8.4 or SW8.4.Mut sequence substituted in the SL3 position of HIV-1 Psi are schematically illustrated. B) Each mutant plasmid was transfected into 293FT cells with packaging plasmids, and the harvested supernatants were determined for viral p24 values and used to transduce MT4 cells. C) Percentage of EGFP-positive MT4 cells from FACS analysis are shown in parenthesis in the figures and the numbers in each figure indicate the relative transducibility of each vector compared to that of pLenti/EGFP transgene vector. D) The viral RNA packaging efficiency was measured by qRT-PCR and normalized to p24 ELISA data. All data are presented in comparison to the EGFP control in One-way ANOVA, and data showing significant differences (p<0.001) are marked with three asterisks.</p

Transduction efficiency and RNA packaging of various lentiviral vectors.

<p>A) One milliliter of lentiviral supernatants harvested from transiently-transfected 293FT cells was used to transduce 1×10⁵ MT4 cells. Expression levels of the EGFP transgene were observed by fluorescence microscopy in 293FT cells and MT4 cells 30 hr post-transfection and 72 hr post-transduction, respectively. B) Amounts of virus particles in p24 in harvested viral supernatants were measured by ELISA. (error bars: ±S.E.M.) C) Percentage of EGFP positive MT4 cells after transduction was determined by FACS as described in Materials and Methods. D) The transducibility of each virus was calculated by normalization with p24 ELISA data, and each graph represents five different experiments performed independently. E) One microliter of viral supernatant was subjected to direct virus lysis and qRT-PCR to measure packaged viral RNA, and the results were normalized with the ELISA data. Statistically different results were tagged with two (p<0.05) or three (p<0.0001) asterisks (One-way ANOVA).</p

The effect of serial deletions of the Psi sequence on gene transduction efficiency and RNA packaging.

<p>A) Psi sequence of PMM.psi vector was deleted from 3′ end serially. The nucleotide position numbers were also shown in parentheses at the end of each mutant constructs. B) Lentiviruses produced from PMM1.psi deletion mutants were transduced to MT4 cells for 72 hr, and expression of EGFP was visualized by fluorescence microscopy and percentage of EGFP positive cells was quantified with FACS and indicated on each histogram. C) Transduction efficiency of each vector in MT4 cells was calculated from FACS data normalized with p24 ELISA. D) Viral RNA packaging efficiency was determined from qRT-PCR and p24 ELISA. All data are presented in comparison to EGFP control in One-way ANOVA, and data showing significant differences (p<0.001) are marked with three asterisks.</p

Schematic representation of lentiviral plasmids used in this study.

<p>A) The numbers in parentheses at the top designated as pLenti/EGFP indicate each region of viral sequence elements residing in the 5′ UTR of HIV-1 according to the genome sequence of HXB2 isolate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050148#pone.0050148-Clever2" target="_blank">[19]</a>. Shown below is an enlargement of the 5′ UTR region and its nucleotide sequence. Black triangles around the packaging sequence Psi indicate nucleotide positions in which restriction enzyme sites were created to generate lentiviral transgene vectors used in this study. Open triangles indicate each nucleotide positions to generate serial deletion mutants of the Psi tested in this study, which are also schematically shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050148#pone-0050148-g006" target="_blank">Figure 6A</a>. B) Nucleotide positions and sequence changes introduced in the transgene vector constructs are indicated.</p

Determination of lentiviral titers and transduction efficiency.

*<p>CFU: Colony Forming Unit.</p